Control of plant virus diseases took a major step forward when it was shown that the tobacco mosaic virus (“TMV”) coat protein (“CP”) gene that was expressed in transgenic tobacco conferred resistance to TMV (Powell-Abel et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science 232:738-43 (1986)). The concept of pathogen-derived resistance (“PDR”), which states that pathogen genes that are expressed in transgenic plants will confer resistance to infection by the homologous or related pathogens (Sanford et al., “The Concept of Parasite-Derived Resistance—Deriving Resistance Genes from the Parasite's Own Genome,” J. Theor. Biol., 113:395-405 (1985)) was introduced at about the same time. Since then, numerous reports have confirmed that PDR is a useful strategy for developing transgenic plants that are resistant to many different viruses (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol., 33:323-43 (1995)).
Remarkable progress has been made in developing virus resistant transgenic plants despite a poor understanding of the mechanisms involved in the various forms of pathogen-derived resistance (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol. 33:323-43 (1995)). Although most reports deal with the use of coat protein genes to confer resistance, a growing number of reports have shown that viral replicase (Golemboski et al., “Plants Transformed with a Tobacco Mosaic Virus Nonstructural Gene Sequence are Resistant to the Virus,” Proc. Natl. Acad. Sci. USA 87:6311-15 (1990)), movement protein (Beck et al., “Disruption of Virus Movement Confers Broad-Spectrum Resistance Against Systemic Infection by Plant Viruses with a Triple Gene Block,” Proc. Natl. Acad. Sci. USA, 91:10310-14 (1994)), NIa proteases of potyviruses (Maiti et al., “Plants that Express a Potyvirus Proteinase Gene are Resistant to Virus Infection,” Proc. Natl. Acad. Sci. USA, 90:6110-14 (1993)), and other viral genes are effective. This led to the conclusion that any part of a plant viral genome may give rise to PDR. Furthermore, the viral genes can be effective in the translatable and nontranslatable sense forms, and less frequently, antisense forms (Baulcombe, “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell 8:1833-44 (1996); Dougherty et al., “Transgenes and Gene Suppression: Telling us Something New?,” Current Opinion in Cell Biology 7:399-05 (1995); Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol. 33:323-43 (1995)).
RNA-mediated resistance is the form of PDR where there is clear evidence that viral proteins do not play a role in conferring resistance to the transgenic plant. The first clear cases for RNA-mediated resistance were reported in 1992 for tobacco etch (“TEV”) potyvirus (Lindbo et al., “Pathogen-Derived Resistance to a Potyvirus Immune and Resistance Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,” Mol. Plant Microbe Interact. 5:144-53 (1992)), potato virus Y (“PVY”) potyvirus (Van Der Vlugt et al., “Evidence for Sense RNA-Mediated Protection to PVY in Tobacco Plants Transformed with the Viral Oat Protein Cistron,” Plant Mol. Biol., 20:631-39 (1992), and for tomato spotted wilt (“TSWV”) tospovirus (de Haan et al., “Characterization of RNA-Mediated Resistance to Tomato Spotted Wilt Virus in Transgenic Tobacco Plants,” Bio/Technology 10:1133-37 (1992)).
Other workers confirmed the occurrence of RNA-mediated resistance with potyviruses (Smith et al., “Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,” Plant Cell 6:1441-53 (1994)), potexviruses (“PXV”) (Mueller et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal 7:1001-13 (1995)), and TSWV and other tospoviruses (Pang et al., “Resistance of Transgenic Nicotiana Benthamiana Plants to Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses: Evidence of Involvement of the N Protein and N Gene RNA in Resistance,” Phytopathology 84:243-49 (1994); Pang et al., “Different Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt Virus and Impatiens Necrotic Spot Tospoviruses,” Bio/Technology 11:819-24 (1993)). More recent work has shown that RNA-mediated resistance also occurs with the comovirus cowpea mosaic virus (Sijen et al., “RNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,” Plant Cell 8:2227-94 (1996)) and squash mosaic virus (Pang et al., “Resistance to Squash Mosaic Comovirus in Transgenic Squash Plants Expressing its Coat Protein Genes,” Molecular Breeding 6:87-93 (2000)).
Major advances towards understanding the mechanism(s) of RNA-mediated resistance were made by Dougherty and colleagues in a series of experiments with TEV and PVY. Using TEV, this group showed that transgenic plants expressing translatable full length coat protein, truncated translatable coat protein, antisense coat protein genes, and nontranslatable coat protein genes had various phenotypic reactions after inoculation with TEV (Lindbo, J. A., “Pathogen-Derived Resistance to a Potyvirus Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,” Mol. Plant Microbe Interact. 5:144-53 (1992) and Lindbo et al., “Untranslatable Transcripts of the Tobacco Etch Virus Coat Protein Gene Sequence Can Interfere with Tobacco Etch Virus Replication in Transgenic Plants and Protoplasts,” Virology 189:725-33 (1992)). Transgenic plants displayed resistance, recovery (inoculated plants initially show systemic infection but younger leaves that develop later are symptomless and resistant to the virus), or susceptible phenotypes. Furthermore, they showed that leaves of resistant plants and asymptomatic leaves of recovered plants had relatively low levels of steady state RNA when compared to those in leaves of susceptible plants (Lindbo et al., “Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,” Plant Cell 5:1749-59 (1993)). However, nuclear run off experiments showed that those plants with low levels of steady state RNA had higher transcription rates of the viral transgene than those plants that were susceptible (and had high steady state RNA levels). To account for these observations, it was proposed “that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation” (Lindbo et al., “Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,” Plant Cell 5:1749-59 (1993)). It was also suggested that the low steady state RNA levels may be due to post-transcriptional gene silencing (“PTGS”), causing a lack of expression of the transcribed gene, a phenomenon that was first proposed by de Carvalho et al., “Suppression of Beta-1,3-glucanase Transgene Expression in Homozygous Plants,” EMBO J. 11:2595-602 (1992) for the suppression of β-1,3-glucanase transgene in homozygous transgenic plants.
An RNA threshold model was proposed to account for these observations (Lindbo et al., “Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,” Plant Cell 5:1749-59 (1993)). This model states that there is a cytoplasmic cellular degradation mechanism that acts to limit the RNA levels in plant cells, and that this mechanism is activated when the transgenic RNA transcript goes above a threshold level. The degradation mechanism is specific for the transcript that goes above the threshold level; and if the transcript that goes above a certain threshold is a viral transgene, the virus resistance state is observed in the plant, because the degradation mechanism also targets, for inactivation, the specific sequences of the incoming virus. The model also accounts for the ‘recovery’ of transgenic plants by suggesting that viral RNA from the systemically invading virus triggers the phenomenon in some transgenic plants that have two copies of the transgenes. Plants that had more than three copies of the transgenes caused the threshold level to be surpassed without the invasion of virus (Goodwin et al., “Genetic and Biochemical Dissection of Transgenic RNA-Mediated Virus Resistance,” Plant Cell 8:95-105 (1996); Smith et al., “Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,” Plant Cell 6:1441-53 (1994)). Although the degradation mechanism is not clear, it is proposed that a cellular RNA dependent RNA polymerase (“RdRp”) binds to the transcript and produces small fragments of antisense RNA which then bind to other transcripts to form duplexes which are then degraded by nucleases that specifically recognize RNA-RNA duplexes. This degradation mechanism is sequence specific, which accounts for the specificity of RNA-mediated resistance.
Work on PVX by Baulcombe and colleagues (English et al., “Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes,” Plant Cell 8: 179-88 (1996); Mueller et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal 7:1001-13 (1995)) confirmed and extended the results by Dougherty and colleagues. An aberrant RNA model which is a modification of the RNA threshold model of Dougherty was proposed. The features of the model are similar to the Dougherty model except that it states that the RNA level is not the sole trigger to activate the cellular degradation mechanism, but instead aberrant RNAs that are produced during the transcription of the transgene play an important part in activating the cytoplasmic cellular mechanism that degrades specific RNA. The production of aberrant RNA may be enhanced by positional affects of the transgene on the chromosome and by methylation of the transgene DNA. The precise nature of the aberrant RNA is not defined, but it may contain a characteristic that makes it a preferred template for the production of antisense RNA by the host encoded RdRp (Baulcombe, D. C., “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell 8:1833-44 (1996); English et al., “Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes,” Plant Cell 8: 179-88 (1996)). Thus, the model also proposes that RdRp and antisense molecules are involved in the degradation mechanism. Baulcombe and colleagues confirmed that plants which show low steady state transgene levels have multiple copies of transgenes and that the low steady state RNA and the accompanying resistant state is due to post-transcriptional gene silencing. The term homology-dependent resistance was proposed to describe the resistance in plants that show homology-dependent gene silencing (Mueller et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal 7:1001-13 (1995)).
Numerous reports have been published on critical advances in the understanding of the biochemistry and genetics of both gene silencing and RNA-interference. Similarities between RNA-interference (“RNAi”) and post-transcriptional gene silencing are astonishing, and point all to the crucial role played by sequence homology in triggering these two mechanistically related phenomena (Matzke et al., “RNA-Based Silencing Strategies in Plants,” Curr. Opin. Genet. Dev. 11(2):221-227 (2001)). In RNAi, the introduction of double stranded RNA into animal or plant cells leads to the destruction of the endogenous, homologous mRNA, phenocopying a null mutant for that specific gene. In both post-transcriptional gene silencing and RNAi, the dsRNA is processed to short interfering molecules of 21-,22- or 23-nucleotide RNAs (“siRNA”) by a putative RNAaseIII-like enzyme (Tuschl T., “RNA Interference and Small Interfering RNAs,” Chembiochem 2: 239-245 (2001); Zamore et al., “RNAi: Double Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals,” Cell 101, 25-3, (2000)). The endogenously generated siRNAs mediate and direct the specific degradation of the target mRNA. In the case of RNAi the cleavage site in the mRNA molecule targeted for degradation is located near the center of the region covered by the siRNA (Elbashir et al., “RNA Interference is Mediated by 21- and 22-Nucleotide RNAs,” Gene Dev. 15(2):188-200 (2001)).
Whether the same model applies for post-transcriptional gene silencing is still under debate (however, see Thomas et al., “Size Constraints for Targeting Post-Transcriptional Gene Silencing and for RNA-Directed Methylation in Nicotiana Benthamiana Using a Potato Virus X Vector,” Plant J. 25(4):417-425 (2001)).
Tomato Spotted Wilt Virus (“TSWV”) is a very damaging virus of worldwide distribution that attacks ornamentals and vegetable crops, causing multimillion-dollar losses annually. TSWV belongs to the Tospoviridae family (Bunyavirus group), has a tripartite genome composed of sense and antisense RNA, and is transmitted by thrips in a persistent manner. Tobacco plants have been engineered to express full or partial version of the nucleocapsid (“N”) gene of TSWV-BL. It has been clearly demonstrated that any single fragment of the TSWV-BL N gene is able to confer resistance against the virus by post-transcriptional gene silencing (Pang et al., “Nontarget DNA Sequences Reduce the Transgene Length Necessary for RNA-Mediated Tospovirus Resistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997)). Moreover, the fragments can be reduced to a minimum of 100 nt long and still trigger post-transcriptional gene silencing if transcriptionally fused to a non-related, carrier DNA (Pang et al., “Nontarget DNA Sequences Reduce the Transgene Length Necessary for RNA-Mediated Tospovirus Resistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997) (“Pang 1997)). Furthermore, it has been shown that the use of short fragments allowed the incorporation of viral gene fragments from multiple viral sources, imparting resistance to the plant against a plurality of viral pathogens (Jan, Doctor in Philosophy Thesis Dissertation, “Roles of Non-Target DNA and Viral Gene Length in Influencing Multi-virus Resistance Through Homology-Dependent Gene Silencing,” Cornell University, p. 286 (1988)). These short fragments, which individually have insufficient length to impart such resistance, are more easily and cost effectively produced than full length genes. Furthermore, there is no need to include in the plant separate promoters for each of the fragments; only a single promoter is required.
Two important, and heretofore unanswered questions related to genetically engineered viral resistance using short viral DNA fragments are: 1) how do changes in sequence homology of the transgene affect its effectiveness in conferring resistance, and 2) can a synthetic DNA be produced with sufficient sequence homology such that the synthetic DNA would confer resistance against multiple viruses. While great strides have been made in PDR methodology, such as imparting resistance to multiple viral pathogens, even Pang's method involves time-consuming and expensive steps required to isolate and manipulate multiple viral DNAs for transformation purposes. What is needed now is a method for utilizing sequence homology information to design and create a single, short synthetic transgene that will impart multiple traits, thereby significantly reducing the labor and materials currently invested in cloning and subcloning procedures directed to imparting pathogen resistance and other traits.
The present invention is directed to overcoming these and other deficiencies in the art.